The efficacy of conventional antibiotics has declined in recent years due to the progressive increase and proliferation of antibiotic-resistant organisms (Davies, 1994; Schutze et al., 1994). The discovery of a large number of naturally occurring invertebrate and vertebrate antimicrobial peptides has resulted in the emergence of novel classes of peptide antibiotics that exhibit remarkable selectivity for prokaryotes and minimize problems of introducing microbial resistance (Boman, 1998; Hancock & Lehrer, 1998; Hancock & Chapple, 1999; Nizet et al., 2001; Zasloff, 2002). These peptide antibiotics interact directly with microbial surfaces, often leading to the formation of pores or in some way compromising membrane permeability (Zasloff, 1992; Hancock, 1997a; Hancock & Rozek, 2002; Koczulla & Bals, 2003; Yeaman & Yount, 2003). Such molecules exhibit diverse structures; however, most are cationic amphiphilic molecules because of the presence of arginine and lysine residues and can be classified into four or five different structural groupings. These include: (a) cysteine-rich, amphiphilic β-sheet peptides (α- and β-defensins, protegrins, and tachyplesins); (b) cysteine-disulfide ring peptides with or without amphiphilic tails (bactenecin, ranalexin, and brevinins); (c) amphiphilic α-helical peptides without cysteine (magainins and cecropins); and (d) linear peptides with one or two predominant amino acids (proline or tryptophan) (Hancock et al., 1995; Hancock, 1997b; Hancock & Lehrer, 1998; Henderson et al., 1998).
Many synthetic analogs to these peptides have been created in attempts to improve the antimicrobial activity of some of these naturally occurring antibacterial peptides (Wade et al., 1992, Tamamura et al., 1995; Helmerhorst et al., 1997; Fuchs et al., 1998; Chen et al., 2000; Mosca et al., 2000; Rothstein et al., 2001). For example, Dhvar 5, an analog of histatin 5, one of the antimicrobial histatin peptides that are derived from saliva (Helmerhorst et al., 1999; Mickels et al., 2001), and IB-367, an analog of protegrins, the antimicrobial peptides that were isolated form porcine leukocytes (Zhao et al., 1994; Chen et al., 2000; Mosca et al., 2000), are more effective in inhibiting bacterial growth and are easily synthesized as compared to their native counterparts. Although, new antimicriobial peptides have been developed, many of these peptides are large, complex, and are difficult and expensive to synthesize. Accordingly, a need exists for more effective and broad-spectrum antimicrobial peptides that are more easily synthesized. In particular, a need exists for an antimicrobial peptide that is effective in inhibiting the growth of various microorganisms that exist in the human mouth. In addition, a need exists for a peptide that is effective in preventing the formation of biofilms, which have been implicated in the development of infections and diseases such as gingivitis and various forms of periodontal diseases. In general, biofilms may include microorganisms such as bacteria, fungi, yeast, viruses and protozoa.
Typically, biofilms are not structurally homogeneous monolayers of microbial cells on a surface. Rather, they can be described as heterogeneous. Living, fully hydrated biofilms are composed of cells and matrix material, wherein the cells are located in matrix-enclosed “towers” and “mushrooms”. Once a biofilm has formed and the matrix has been secreted by the sessile cells, the resultant structure is highly viscoelastic and behaves in a rubbery manner. (Donlan et al., 2002).
Biofilms form preferentially at high-shear locations in natural and industrial systems. Smooth surfaces are colonized just as easily as rough surfaces and the physical characteristics of a surface influence bacterial adhesion to only aminor extent. When biofilms are formed in low-shear environments, they have a low tensile strength and break easily, but biofilms formed at high shear locations are remarkedly strong and resistant to mechanical breakage. (Donlan et al., 2002).
The nature of biofilm structures and the physiological attributes of biofilm organisms confer an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. Mechanisms responsible for resistance may be one or more of the following: i) delayed penetration of the antimicrobial agent through the biofilm matrix, ii) altered growth rate of biofilm organisms, and iii) other physiological changes due to the biofilm mode of growth. For example, antimicrobial molecules must diffuse through the biofilm matrix in order to inactivate the encased cells. The extracellular polymeric substances constituting this matrix present a diffusional barrier for these molecules by influencing either the rate of transport of the molecule to the biofilm interior or the reaction of the antimicrobial material with the matrix material. Due to the strong resistance to antimicrobial agents that biofilms possess, it is essential to prevent the formation of biofilms before they can cause harm in an environment. (Donlan et al., 2002).
As described above, a biofilm is a complex, highly differentiated, multicultural community that has been shown to cause a variety of infections. The organisms responsible, the extracellular components of the biofilm, the nature of the required conditioning film, and the mode of pathogenicity vary from one disease condition to the next. In most cases, however, there are certain underlying processes that are unchanging: production of an extracellular matrix polymer, resistance to antimicrobial agents that increases with biofilm age, and resistance to immune system clearance. Examples of infections that biofilms have been associated with include, but are not limited to periodontitis, native valve endocarditis, otitis media, chronic bacterial prostatitis, and cystic fibrosis. (Donlan et al., 2002).
Periodontitis is one of the most prevalent infectious diseases in the world, affecting approximately forty-nine million people in the United States alone and 10-12% of the population in the industrialized countries. The following organisms have been isolated from patients with moderate periodontal disease and positively correlated with gingivitis: Porphyromonas gingivalis; Tanarella forsythensis; Treponema denticola; Actinobacillus actinomycetemcomitans; Fusobacterium nucleatum, Peptostreptococcus micros, Eubacterium timidum, Eubacterium brachy, Lactobacillus spp., Actinomyces naeslundii, Pseudomonas anaerobius, Eubacterium sp. strain D8, Prevotella intermedia, Fusobacterium sp., Selenomonas sputigena, Eubacterium sp. strain D6, Bacteroides pneumosintes, and Haemophilus aphrophilus. (Donlan et al., 2002; Socransky et al., 1998).
Proteinaceous conditioning films, called acquired pellicle, develop on exposed surfaces of tooth enamel almost immediately after cleaning of the tooth surface within the oral cavity. The pellicle comprises albumin, lysozyme, glycoproteins, phospoproteins, lipids, and gingival crevice fluid. Within hours of pellicle formation, single cells of primarily gram-positive cocci and rod-shaped bacteria from the normal oral flora colonize these surfaces. The pioneer species are predominantly streptococci, actinomycetes, and smaller numbers of Haemophilus. These organisms have the ability to bind directly to the pellicle through surface proteinaceous appendages and the production of extracellular glucans. After several days, actinomycetes predominate, and the characteristic polysaccharide matrix of a biofilm begins to develop. (Donlan et al., 2002; Marsh et al., 1995).
Organisms associating with and attaching to cells in this early biofilm do so by a process called coaggregation. Coaggregation is cell-to-cell recognition whereby organisms in the biofilm can recognize and adhere to genetically distinct bacteria by means of adhesions. These adhesions recognize protein, glycoprotein, or polysaccharide receptors on oral surfaces, including other cell types. A climax biofilm community, termed plaque, will devlop within 2 to 3 weeks if the plaque is left undisturbed, with 50 to 100 μm thick bioflims developing. However, it is possible for plaque to form within 24 hours. In addition to matrix polysaccharides, there will be polymers of salivary origin. (Donlan et al., 2002; Kolenbrander et al., 1999).
Plaque that becomes mineralized with calcium and phosphate ions is termed calculus or tartar. In addition to development on the tooth surfaces (within fissures), plaque can develop more extensively in protected areas, including approximal areas (between the teeth) and the gingival crevice (between the tooth and gum). As the plaque mass increases in these protected areas, the beneficial buffering and antimicrobial properties of the saliva are less able to penetrate and protect the tooth enamel, leading to dental caries or periodontal disease. (Donlan et al., 2002).
As the organisms develop biofilms in the subgingival crevice, they produce proteolytic enzymes that damage tissue directly or interfere with host defenses. Collangenase and hyaluronidase are also present and capable of degrading collagen. Breakdown of the fiber barrier system may occur, and the lesion may then progress to one that may attack the supporting structures of the tooth. Gram-negative organisms also produce endotoxins that may result in inflammation. It has been demonstrated that the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia are capable of invading epithelium cells in a laboratory assay, eliciting invasion mechanisms similar to those of other pathogens. (Donlan et al., 2002).